Education and microbicides active against HIV represent the best approaches to controlling the epidemic worldwide in the absence of a protective vaccine. Our research program studies the earliest events in genital tract transmission. We have identified a protein expressed by genital tract epithelial cells that could serve as a potential target for inhibition of transmission of HIV called gp-340. We have demonstrated that gp-340 is expressed on the cell surface of vaginal and cervical epithelial cells, in vivo, in vitro, and ex vivo and binds HIV envelope. Of significance to genital tract transmission, gp-340 binding of virus leads to an increase in both the infectivity and half-life of the virus. Gp-340 expressed by genital tract tissue and cell lines also mediates transcytosis of HIV, the vesicular transport of macromolecules from one side of a cell to the other. A second molecule called syndecan has been studied and shown to have similar trans-infection and transcytosis properties and is also expressed by genital tract cells. We have identified a peptide inhibitor of envelope binding to gp-340 that blocks both gp-340 mediated trans-infection and transcytosis in in vitro and ex vivo models of genital tract transmission. This peptide contains a portion of a motif that inhibits syndecan mediated transinfection, as well, and we will modify this peptide to inhibit envelope binding to both macaque gp340 and syndecan and develop it into a microbicide. This potential role of gp-340 and syndecan to act at a stage of infection after delivery to the lumen of the genital tract but prior to interaction with and infection of target cells is very attractive and novel in microbicide design. We hypothesize that interfering with this process will inhibit or block genital tract transmission. In the initial R21 portion of this proposal, we will establish in vitro macaque systems of genital tract transmission. If we demonstrate that macaque gp340 and syndecan mediate trans- infection and transcytosis and V3 loop derived peptides or improved versions block macaque gp340 and syndecan mediated transinfection and transcytosis, we will proceed with the R33 portion of the grant. The specific aims of this are: microbicide development with in vitro testing and to test the effect of blocking gp340 and syndecan-HIV Env interaction on genital tract SIV transmission in the rhesus macaque vaginal transmission model. Through these specific aims, we will develop a new type of microbicide and determine the role of genital tract gp-340 and syndecan in HIV transmission. If successful, these studies will deliver a new microbicide based on host cell interactions with HIV that promote genital tract transmission to preclinical trial studies.